This invention relates to probes comprising enzyme labels and specific binding members such as antibodies and single-stranded nucleic acids, and assays employing such probes.
The use of enzymes as labels in a wide variety of clinical, veterinary and environmental diagnostic assays including enzyme immunoassays and nucleic acid probe-based assays is well known. One example of the use of these employs a sandwich format in which an immobilized antibody, antigen or nucleic acid is used to recognize and bind to a portion of the molecule to be detected. An appropriate enzyme-labelled antibody or nucleic acid probe is then introduced which binds to a different portion of the complex to be measured. This results in the formation of a complex immobilized to the solid surface which is labelled with the enzyme. After several washing steps to remove all traces of the original sample and the excess unbound labelled moiety, a substrate for the enzyme is introduced, and the presence of the enzyme detected by its action on a substrate to produce a change in colour, fluorescence, redox state, or to produce light.
It is important that enzymes employed as labels catalyze a reaction which has an easily detectable product, and have a high turnover number to allow sensitive detection: horseradish peroxidase and alkaline phosphatase are most common. Although sensitive chemiluminometric assays for horseradish peroxidase have been described which allow small amounts of enzyme to be detected, problems associated with its use include lack of enzyme and substrate stability and the presence of endogenous peroxidases in samples.
For alkaline phosphatase, enzyme amplification cycles have been described which further reduce the amount of enzyme which can be detected, thereby extending the detection limit. For example, in U.S. Pat. No. 5,445,942 to Rabin et al., entitled xe2x80x9cAmplification Assay for hydrolase enzymesxe2x80x9d, a method is described for detecting a hydrolase enzyme able to hydrolyze a synthetic derivative of FAD substituted in such a way that it yields FAD when hydrolyzed. The FAD produced forms an active holoenzyme from the corresponding apoenzyme. This approach allows the detection of small amounts of alkaline phosphatase in short periods of time. For example, we have used such an amplification system in which the apoenzyme is apo-D-amino acid oxidase to measure 0.1 amol of alkaline phosphatase in less than 30 minutes (Harbron S, et al., Anal. Biochem. (1992) 206: 119-124).
However, the use of alkaline phosphatase as the label enzyme has a number of shortcomings: its large size (MW=140,000) means that it can sterically hamper the association of the antibody or nucleic acid probe with its target; its nature as a membrane-associated protein means that it binds non-specifically to many surfaces; it is very sensitive to the presence of phosphate carried over from previous assay stages; it has limited stability at the temperatures often used in nucleic acid hybridization steps; and it is a commonly occurring enzyme in many tissues and occurs in the environment at large as a component of bacteria and other microorganisms. Rabin et al. describe the use of the amplification assay for the detection of sulphatases, carboxylesterases, acetylesterase and venom phosphodiesterase which may obviate some of these problems, but they do not teach that the approach could be used for the assay of enzymes of the nuclease class, such as nuclease S1 and nucleate P1. It is known that nuclease P1 hydrolyses Coenzyme A (Fujimoto et al., Agr. Biol, Chem. (1974) 38: 1555-1561).
EP-A-401,001 concerns novel dioxetanes having a substituent -X-Y-Z where Z and Y are protecting groups which are removable successively, leading to chemiluminescence. For a sandwich assay, Z may be removed by a first triggering enzyme E1 which is directly or indirectly bound to an antigen, antibody or nucleic acid probe. E1 may be a nuclease.
Further examples of assays involving enzyme-containing probes are provided by EP-A-0,304,934, U.S. Pat. No. 5,563,063, WO-A-96/41015, WO-A-90/00252, EP-A-0,061,071, EP-A-0,124,124, EP-A-0,516,948 and GB-A-2018986.
We have discovered that both nuclease S1 and nuclease P1 can hydrolyze the synthetic analogue of FAD in which the 3xe2x80x2 hydroxyl group on the ribose moiety of FAD is esterified with phosphoric acid to give 3xe2x80x2 FADP, thereby giving a new means of assaying these enzymes in an extremely rapid and sensitive fashion. But Fujimoto et al. also showed that nuclease P1 hydrolyses single stranded DNA and RNA, which would indicate that this enzyme is unsuitable for labelling nucleic acid probes. In fact, the prior art teaches that nucleases are used for degrading nucleic acids: thus U.S. Pat. No. 5,145,780, to Oishi and Aoi describes an enzyme preparation produced by a fungus such as Trichoderma, Aspergillus and Fusarium which contains a nuclease that is active even after heating at 100xc2x0 C. for 30 minutes. This enzyme preparation may be effectively used when it is necessary to decompose nucleic acids at elevated temperature over a prolonged period. U.S. Pat. No. 5,006,472, to Dove and Mitra, discloses a method for purifying rDNA or monoclonal antibody culture products by using nuclease enzyme treatment to degrade undesirable residual nucleic acids to a molecular size or charge range sufficiently different from the product to be purified so that this difference can be exploited in a subsequent purification step (e.g. precipitation, size exclusion chromatography or ion exchange chromatography).
Although it would not therefore be expected that nuclease P1 and nuclease S1 could be used to label nucleic acids, Fujimoto et al. demonstrated that the ability of nuclease P1 to hydrolyze single-stranded nucleic acids was pH dependent, and we have found that pH values greater than 7.0 allow the labelling of nucleic acids with these nucleases.
Broadly, the present invention relates to the use of P1 and S1 nucleases as enzyme labels for assays. Thus in one aspect the invention provides a probe which comprises a nuclease (particularly P1 or S1) coupled to a specific binding member (xe2x80x9csbmxe2x80x9d) (generally an antibody or a single-stranded nucleic acid). The nuclease is preferably covalently attached to the sbm.
In another aspect the invention provides a method of producing a probe which comprises coupling a nuclease to an sbm.
In further aspects the invention provides an assay method employing a probe according to the first aspect, and a kit for carrying out such an assay.
A preferred type of sbm is antibodies (particular IgG antibodies) and functional fragments thereof capable of binding to a target in an assay procedure.
Another preferred type of sbm is nucleic acids (DNA, RNA or analogues thereof), generally oligonucleotides. The nucleic acid may be produced with a derivatised 5xe2x80x2-end (e.g. trityl-hexyl thiol derivatised) to facilitate coupling to a nuclease which has been rendered susceptible to disulphide exchange, e.g. being 2-pyridyl disulphide activated.
Preferred embodiments of the invention may enable one to achieve one or more of the following objects and advantages:
(a) to provide an enzyme label which is small and which does not interfere with the association of antibody and antigen, nor of complementary strands of nucleic acid;
(b) to provide an enzyme label which may be easily conjugated to antibodies and nucleic acids using well-known methodologies;
(c) to provide an enzyme label which is not membrane associated in its natural state, and which is secreted into the growth medium, and which therefore has a low level of non-specific binding to solid surfaces;
(d) to provide an enzyme label which is largely insensitive to the presence of phosphate, allowing it to be used in automated assay machinery in which phosphate-containing washing solutions are routinely used;
(e) to provide an enzyme label which has good temperature stability, allowing it to be used at high temperature, particularly in nucleic acid assays;
(f) to provide an enzyme label which is not a commonly occurring enzyme, thereby avoiding contamination from endogenous enzyme in the sample.
Further objects and advantages are to provide the use as enzyme labels of enzymes which are commercially available, which are not inhibited by phosphate monoesters which may be included in the assay solution to prevent endogenously occurring phosphatases from hydrolysing 3xe2x80x2 FADP, which do not hydrolyze single-stranded nucleic acids at the pH employed in the assay solution, and which can be assayed using an enzyme amplification system.
Some embodiments of the invention will be described in more detail, by way of example, with reference to the accompanying drawings.